Richard A.J. Darby

Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

BackgroundThe production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts.ResultsWe show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis.ConclusionThis work demonstrates the power of a rational approach to recombinant protein production by using the results of transcriptome analysis to engineer improved strains, thereby revealing the underlying biological events involved.

Which yeast species shall I choose? Saccharomyces cerevisiae versus Pichia pastoris (review)

Having decided on yeast as a production host, the choice of species is often the first question any researcher new to the field will ask. With over 500 known species of yeast to date, this could pose a significant challenge. However, in reality, only very few species of yeast have been employed as host organisms for the production of recombinant proteins. The two most widely used, Saccharomyces cerevisiae and Pichia pastoris, are compared and contrasted here.

LacI-mediated sequence-specific affinity purification of plasmid DNA for therapeutic applications

Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence‐specific zinc finger protein. We present a more generically applicable protein‐based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His‐tagged LacI protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80% based on recovered starting material, 35–50% overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity‐based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids.

Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory

BackgroundRecombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions.ResultsHere we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeasts optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory.ConclusionsThe accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory S. cerevisiae strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to resort to complex control or cultivation schemes. This is anticipated to be of particular value in the production of challenging targets such as membrane proteins.

Understanding the yeast host cell response to recombinant membrane protein production

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industrys strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the proteins native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

Optimising Pichia pastoris Induction

A common method for inducing the production of recombinant proteins in Pichia pastoris is through the use of methanol. However, the by-products of methanol metabolism are toxic to yeast cells and therefore its addition to recombinant cultures must be controlled and monitored throughout the process in order to maximise recombinant protein yields. Described here are online and off-line methods to monitor and control methanol addition to bench-top-scale bioreactors.

Disruption of Yeast Cells to Isolate Recombinant Proteins

Yeast is a proven host for the production of recombinant proteins, which may be incorporated in cellular membranes or localized in subcellular compartments. In order to gain access to these proteins, cellular disruption is required to permit extraction, purification, and downstream analysis. Disruption can significantly impact the yield and quality of the biomaterial. We highlight several disruption techniques that are applicable to yeast cells ranging from mechanical to nonmechanical approaches. In all cases fast, efficient cellular disruption is desirable, that does not alter the protein chemically or physically and that generates material for downstream purification and analysis.

Yeast Transformation to Generate High-Yielding Clones

There are several ways to introduce non-native DNA into yeast cells, including chemical transformation and electroporation. Methods for both of these procedures are outlined in this chapter. Both methods permit the uptake of DNA from the environment through yeast cell membranes and this DNA can be episomally maintained or integrated into the host genome. However, yeast cells must first be made competent to permit passive entry of the DNA and various methods are outlined in this chapter to facilitate this. All of the described methods can be applied in combination with antibiotic or auxotrophic selection pressure.

Production of membrane proteins in yeast

Background Yeast is an important and versatile organism for studying membrane proteins. It is easy to cultivate and can perform higher eukaryote-like post-translational modifications. S. cerevisiae has a fully-sequenced genome and there are several collections of deletion strains available, whilst P. pastoris can produce very high cell densities (230 g/l). Results We have used both S. cerevisiae and P. pastoris to over-produce the following His6 and His10 carboxyl terminal fused membrane proteins. CD81 – 26 kDa tetraspanin protein (TAPA-1) that may play an important role in the regulation of lymphoma cell growth and may also act as the viral receptor for Hepatitis C-Virus. CD82 – 30 kDa tetraspanin protein that associates with CD4 or CD8 cells and delivers co-stimulatory signals for the TCR/CD3 pathway. MC4R – 37 kDa seven transmembrane G-protein coupled receptor, present on neurons in the hypothalamus region of the brain and predicted to have a role in the feast or fast signalling pathway. Adt2p – 34 kDa six transmembrane protein that catalyses the exchange of ADP and ATP across the yeast mitochondrial inner membrane. Conclusion We show that yeasts are flexible production organisms for a range of different membrane proteins. The yields are such that future structure-activity relationship studies can be initiated via reconstitution, crystallization for X-ray diffraction or NMR experiments.